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1.
Biotechnol Bioeng ; 116(11): 2852-2863, 2019 11.
Article in English | MEDLINE | ID: mdl-31389000

ABSTRACT

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L -1 ·h -1 ) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L -1 ·h -1 ).


Subject(s)
Caproates/metabolism , Coumarins/metabolism , Escherichia coli/metabolism , Lactones/metabolism , Metabolic Engineering , Catalysis , Escherichia coli/genetics , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Mixed Function Oxygenases/biosynthesis , Mixed Function Oxygenases/genetics
2.
Chembiochem ; 12(17): 2560-3, 2011 Nov 25.
Article in English | MEDLINE | ID: mdl-21984076

ABSTRACT

In a spin: Spin-labeled oligonucleotides produced by click chemistry can be studied by EPR, by using a DEER sequence. This was used to test a complex triple-labeling strategy with damaged DNA. Extensive and accurate analysis of DNA structure and enzymatic repair processes were performed after digestion by EndoIV. Modified DNA structures and DNA-protein interactions can now be readily studied.


Subject(s)
DNA/metabolism , Deoxyribonuclease IV (Phage T4-Induced)/metabolism , Spin Labels , Click Chemistry , DNA Cleavage , DNA Damage , Electron Spin Resonance Spectroscopy , Oligonucleotides/chemistry
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